EP0217814A1 - Herstellungsverfahren für menschliches wachstumhormon. - Google Patents
Herstellungsverfahren für menschliches wachstumhormon.Info
- Publication number
- EP0217814A1 EP0217814A1 EP86901361A EP86901361A EP0217814A1 EP 0217814 A1 EP0217814 A1 EP 0217814A1 EP 86901361 A EP86901361 A EP 86901361A EP 86901361 A EP86901361 A EP 86901361A EP 0217814 A1 EP0217814 A1 EP 0217814A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- hgh
- glu
- amino acid
- ala
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/545—IL-1
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
Definitions
- the present invention concerns a process for producing human growth hormone (hGH) from amino terminal extended hGH using Dipeptidyl Aminopeptidase I (DAP I) (E.C. 3.4, 14.1).
- hGH human growth hormone
- DAP I Dipeptidyl Aminopeptidase I
- Pseudomonas aeruginose for the production of hGH without methionine for therapeutic use, however, is vitiated by the risk that this bacterium and many other Pseodomonas bacteria, which- are potentially pathogenic, synthetize toxic toxines which are difficult to remove by purification of hGH.
- the present invention is based on the idea that it is possible to produce biosynthetic hGH which has attached to it a pre-sequence which can be cleaved enzymatically in a high yield, and which gives products by the enzy ⁇ matic cleavage which can be separated satisfactorily by known purification methods, such as chromatography .
- purification methods such as chromatography
- Dipeptidyl aminopeptidase (E.C. 3.4.14.1) is used for cleaving a specific amino terminal extension on hGH.
- This enzyme is specific in that it can cleave dipeptides from the N-terminus of peptides and proteins, i.a. on the following conditions:
- N-terminal amino acid in the protein/peptide is not Lys or Arg.
- the extension must not contain Lys or Arg at the uneven sites, and if Pro occurs, it may only be positioned as an N-terminal amino acid since, otherwise, the cleavage of dipeptides would stop before the entire extension has been cleaved.
- amino acids in the extension which enable analytic and preparative separation of authentic hGH from amino terminal extended hGH. This may be done by selecting amino acids with charged side chains, i.e. Glu or Asp (negatively charged side chains) or Arg, Lys or His (positively charged side chains). Then, by anion exchange and cation exchange (analytic or preparative) or by anionic and cationic electrophoresis it will be possible to separate amino terminal extended hGH from (authentic) hGH - provided that no combination of positively and negatively charged amino acid residues in the extension has been selected which neutralize each other in terms of charge.
- charged side chains i.e. Glu or Asp (negatively charged side chains) or Arg, Lys or His (positively charged side chains).
- anion exchange and cation exchange analytic or preparative
- anionic and cationic electrophoresis it will be possible to separate amino terminal extended hGH from (authentic) hGH - provided that no combination of positively and negatively charged amino acid residue
- At least one of the charged amino acids may advantageously be contained in the dipeptide component, which is attached directly to the N-terminus of hGH, because it may then be observed whether the entire amino terminal ex ⁇ tension has been cleaved.
- the last amino acid residue in the extension is an amino acid with a charged side chain. This is particu ⁇ larly important when the microorganism in vivo only partly cleaves the N-terminal methionine residue.
- an amino acid with charged side chains in the amino terminal extension to hGH is either exclusively positively or negatively charged. This prevents amino terminal extended hGH, partly en ⁇ zymatically converted amino terminal extended hGH and authentic hGH from having the same net charge at any time.
- amino terminal extensions may be obtained by fermenting in a suitable substrate a microorganism transformed with a plasmid, which codes for human growth hormone with these attached amino ter ⁇ minal extensions.
- methionine which is the N-terminal amino acid in all proteins formed in E. coli, is cleaved enzymatically in the microorganism after ex ⁇ pression of the protein. This results e.g. in the above- mentioned amino terminal extended hGH proteins.
- proteins are purified by conventional purifica ⁇ tion methods.
- the amino terminal extension is cleaved selectively and in a high yield, and the formed hGH may then easily be separated from any residues of pa.r ly converted amino terminal extended hGH by anion exchange.
- a leucine aminopeptidase preparation from Sigma was used as the starting material for the production of active enzyme or enzyme complex, the active enzyme being frac ⁇ tionated together with leucine aminopeptidase (LAP).
- the active enzyme may by fractionated from LAP by IE- HPLC (ion exchange HPLC) on a highly soluble monoQ from Pharmacia. Fractionation
- Injection volume 500 ul.
- the leucine aminopeptidase activity in the individual fractions is indicated in the drawing. There is only LAP activity in fractions 13 and 14. A balance calculation on LAP activity shows that all applied LAP activity is eluted in these two fractions.
- a cloned DNA sequence which codes for a protein having an amino acid sequence like human growth hormone, hGH (191 amino acid residues, the first four amino acids of which are Phe-Pro-Thr-lie ) is coupled with the following synthetically produced, dual-stranded DNA sequence so that the 3'end of the + strand is coupled to the +5' end of the above-mentioned gene, and the 5' end of the synthetic DNA sequence strand is coupled to the 3' end of the above-mentioned gene by blunt end ligature
- the above-mentioned gene is introduced by ordinary gene cloning techniques into an expression plasmid containing a fusioned Trp-Lac promotor as well as the SD sequence AGGA.
- This structure should express Met-Ala-Glu-hGH .
- This plasmid structure is then introduced in an E. coli cell by prior art techniques.
- a suitable clone containing the above-mentioned structure is isolated and cultivated in a 5 1 scale.
- the cells were harvested by centrifuga- tion and are suspended in a small volume and lyzated using a so-called "French press".
- the expected fusion protein could be demonstrated in the above-mentioned bacterial extract by immunological methods using hGH antibodies, corresponding to a concentration of 200 mg/1 in the culture medium.
- the fusion protein is purified conventionally by anion exchange, ammonium sul- fate precipitation and hydrophobic chromatography.
- the purified (expected) Met-Ala-Glu-hGH was evaluated to be more than 99 % pure, evaluated by SDS electrophoresis. An amino terminal sequence determination showed that the purified hGH material had the sequence Ala-Glu-hGH, which means that Met has been cleaved by an E. coli enzyme.
- DAP I activity isolated from pork kidneys as stated above.
- the reaction mixture was then incubated at 40 °C. After 4 1/2 hours the mixture was cooled to 4 °C. The cooled reaction mixture was then fractionated by anion exchange,, and following this the main peak (hGH product) was iso ⁇ lated. The yield was 90 % .
- the hGH product was evaluated to be more than 99?. pure, evaluated by SDS electrophoresis.
- An amino terminal deter ⁇ mination (Edman degradation) showed that the amino ter ⁇ minal sequence of the hGH product was Phe-Pho-Thr-Ile- Pro-, i.e. as for authentic hGH.
- the biological activity of the hGH product was determined by a tibia, test and was found to be 2.5 IU/mg, which is also the case with authentic hGH.
- Met-Glu-Ala-Glu-hGH is produced by gene techniques in principle as described in example 1. Met-Glu-Ala-Glu-hGH is purified from the fermentation product by anion ex ⁇ change and hydrophobic interaction chromatography. The purified Met-Glu-Ala-Glu-hGH was evaluated to be more than 99% pure by IE-HPLC and SDS electrophoresis
- the reaction mixture was then incubated at 40 °C for 6-0 minutes, resulting in a more than 98. ⁇ conversion of Met-Glu-Ala-Glu-hGH to hGH.
- the reaction mixture was cooled to 4 °C after completed reaction.
- the further purification comprises isoprecipitation, gel filtration and an anion exchange.
- the hGH product was evaluated to be more than 99? ⁇ pure evaluated by IE-HPLC and SDS electrophoresis.
- An amino terminal sequence determination by Edman degradation showed that the amino terminal sequence of the hGH pro ⁇ duct was Phe-Pro-Thr-Ile-Pro-Leu , i.e. as for authentic hGH.
- the biological activity of the hGH product was deter ⁇ mined by a tibia test and was found to be equipotent with pituitary hGH.
- Met-Ala-Glu-hGH is produced by gene techniques in prin ⁇ ciple as described in example 1. Met is cleaved in vivo so that the protein formed by the fermentation is Ala- Glu-hGH. This is purified conventionally by anion ex- change and hydrophobic interaction chromatography.
- the purified Ala-Glu-hGH was evaluated to be more than 99? ⁇ pure by IE-HPLC and SDS electrophoresis.
- the reaction mixture was incubated at 40 °C for 60 minu ⁇ tes, resulting in a more than 98? ⁇ conversion of Ala-Glu- hGH.
- the reaction mixture was cooled to 4 ⁇ C after com ⁇ pleted reaction.
- the further purification comprises iso ⁇ precipitation, gel filtration and an anion exchange.
- the hGH product was found to be more than 99? ⁇ pure evalu ⁇ ated by IE-HPLC and SDS electrophoresis.
- An amino sequence determination by Ed an degradation showed that the amino terminal sequence of hGH was Phe-Pro-Thr-Ile-Pro- Leu, i.e. as for authentic hGH.
- Met-Phe-Glu-hGH is produced by gene techniques in prin- ciple as described in example 1. Met-Phe-Glu-Glu-hGH is purified from the fermentation product by anion exchange and hydrophobic interaction chromatography.
- the purified Met-Phe-Glu-Glu-hGH was evaluated to be more than 99? ⁇ pure by IE-HPLC and SDS electrophoresis.
- reaction mixture was then incubated at 40 °C for
- the reaction mixture is cooled to 4 °C after completed . reaction.
- the further purifi ⁇ cation comprises isoprecipitation, gel filtration and an anion exchange.
- the hGH product was evaluated to be more than 99? ⁇ pure evaluated by IE-HPLC and SDS electrophoresis.
- An amino terminal sequence determination by Edman degradation showed that the amino terminal sequence of the hGH pro ⁇ duct was Phe-Pro-Thr-Ile-Pro-Leu, i.e. as for authentic hGH.
- the biological activity of the hGH product was deter- mined by a tibia test and was found to be equipotent with pituitary hGH.
- Met-Ala-Glu-Ala-Glu-hGH is produced by gene techniques in principle as described in example 1. Met is cleaved in vivo so that the protein formed by the fermentation is Ala-Glu-Ala-Glu-hGH. This is purified conventionally by anion exchange and hydrophobic interaction chromato- graphy.
- the purified Ala-Glu-Ala-Glu-hGH was evaluated to be more than 99% pure by IE-HPLC and SDS electrophoresis.
- the reaction mixture was incubated at 40 °C for 60 minu ⁇ tes, resulting in a more than 98?_ conversion of Ala-Glu- Ala-Glu-hGH to hGH.
- the reaction mixture was cooled to- 4 °C after completed reaction.
- the further purification comprises isoprecipi ation, gel filtration and an anion exchange.
- the hGH product was found to be more than 99.0 pure evalu ⁇ ated by IE-HPLC and SDS electrophoresis.
- An amino termi ⁇ nal sequence determination by Edman degradation showed that the amino terminal sequence of hGH was Phe-Pro-Thr- Ile-Pro-Leu, i.e. as for authentic hGH.
- the biological activity of the hGH product was deter ⁇ mined by a tibia test and was found to be equipotent with pituitary hGH.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT86901361T ATE52807T1 (de) | 1985-02-07 | 1986-02-06 | Herstellungsverfahren fuer menschliches wachstumhormon. |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK055685A DK55685A (da) | 1985-02-07 | 1985-02-07 | Enzym eller enzymkompleks med proteolytisk aktivitet |
DK556/85 | 1985-02-07 | ||
PCT/DK1986/000014 WO1986004609A1 (en) | 1985-02-07 | 1986-02-06 | A process for producing human growth hormone |
Publications (3)
Publication Number | Publication Date |
---|---|
EP0217814A1 true EP0217814A1 (de) | 1987-04-15 |
EP0217814B1 EP0217814B1 (de) | 1990-05-16 |
EP0217814B2 EP0217814B2 (de) | 1994-12-21 |
Family
ID=8094869
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP86901361A Expired - Lifetime EP0217814B2 (de) | 1985-02-07 | 1986-02-06 | Herstellungsverfahren für menschliches wachstumhormon |
Country Status (7)
Country | Link |
---|---|
US (2) | US20010018199A1 (de) |
EP (1) | EP0217814B2 (de) |
JP (1) | JPH0665318B2 (de) |
AU (1) | AU590258B2 (de) |
DE (1) | DE3671245D1 (de) |
DK (1) | DK55685A (de) |
WO (1) | WO1986004609A1 (de) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE122004000009I1 (de) * | 1984-08-16 | 2004-11-18 | Savient Pharmaceuticals Inc | Methode zur Herstellung von menschlichen Wachstumshormonen. |
IT1228925B (it) * | 1987-08-07 | 1991-07-10 | Eniricerche Spa | Procedimento per la preparazione dell'ormone della crescita umano |
IT1223577B (it) * | 1987-12-22 | 1990-09-19 | Eniricerche Spa | Procedimento migliorato per la preparazione dell'ormone della crescita umano naturale in forma pura |
US5126249A (en) * | 1989-05-09 | 1992-06-30 | Eli Lilly And Company | Enzymatic removal of a protein amino-terminal sequence |
DE4105480A1 (de) * | 1991-02-21 | 1992-08-27 | Boehringer Mannheim Gmbh | Verbesserte aktivierung von rekombinanten proteinen |
TW257792B (de) * | 1992-10-01 | 1995-09-21 | Lilly Co Eli | |
KR970010135B1 (ko) * | 1993-06-17 | 1997-06-21 | 주식회사 엘지화학 | 신규 아미노펩티다제 |
US5573923A (en) * | 1993-12-22 | 1996-11-12 | Eli Lilly And Company | Method for removing N-terminal dipeptides from precursor polypeptides with immobilized dipeptidylaminopeptidase from dictyostelium discoideum |
WO1995030685A1 (en) * | 1994-05-09 | 1995-11-16 | Unizyme Laboratories A/S | An enzymatic process for producing a desired protein from an amino terminal extended protein |
US5614379A (en) * | 1995-04-26 | 1997-03-25 | Eli Lilly And Company | Process for preparing anti-obesity protein |
US5840517A (en) * | 1995-04-26 | 1998-11-24 | Eli Lilly And Company | Process for preparing obesity protein analogs |
DE60232880D1 (de) | 2001-05-24 | 2009-08-20 | Neuren Pharmaceuticals Ltd | Gpe-analoga und peptidomimetika |
US7714020B2 (en) | 2001-05-24 | 2010-05-11 | Neuren Pharmaceuticals Limited | Treatment of non-convulsive seizures in brain injury using G-2-methyl-prolyl glutamate |
US7605177B2 (en) * | 2001-05-24 | 2009-10-20 | Neuren Pharmaceuticals Limited | Effects of glycyl-2 methyl prolyl glutamate on neurodegeneration |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL60184A (en) * | 1979-05-31 | 1984-05-31 | Schering Ag | Process for the specific cleavage of protein sequences from proteins |
US4342832A (en) * | 1979-07-05 | 1982-08-03 | Genentech, Inc. | Method of constructing a replicable cloning vehicle having quasi-synthetic genes |
US4769326A (en) * | 1980-02-29 | 1988-09-06 | The Regents Of The University Of California | Expression linkers |
US4775622A (en) * | 1982-03-08 | 1988-10-04 | Genentech, Inc. | Expression, processing and secretion of heterologous protein by yeast |
US4532207A (en) * | 1982-03-19 | 1985-07-30 | G. D. Searle & Co. | Process for the preparation of polypeptides utilizing a charged amino acid polymer and exopeptidase |
US4745069A (en) * | 1982-05-25 | 1988-05-17 | Eli Lilly And Company | Cloning vectors for expression of exogenous protein |
ATE30246T1 (de) * | 1982-12-10 | 1987-10-15 | Nordisk Gentofte | Verfahren zur herstellung reifer proteine aus schmelzproteinen hergestellt in pro- oder eukaryotischen zellen. |
US4755465A (en) * | 1983-04-25 | 1988-07-05 | Genentech, Inc. | Secretion of correctly processed human growth hormone in E. coli and Pseudomonas |
DE122004000009I1 (de) * | 1984-08-16 | 2004-11-18 | Savient Pharmaceuticals Inc | Methode zur Herstellung von menschlichen Wachstumshormonen. |
US4865974A (en) * | 1985-09-20 | 1989-09-12 | Cetus Corporation | Bacterial methionine N-terminal peptidase |
-
1985
- 1985-02-07 DK DK055685A patent/DK55685A/da not_active Application Discontinuation
-
1986
- 1986-02-06 WO PCT/DK1986/000014 patent/WO1986004609A1/en active IP Right Grant
- 1986-02-06 EP EP86901361A patent/EP0217814B2/de not_active Expired - Lifetime
- 1986-02-06 JP JP61501206A patent/JPH0665318B2/ja not_active Expired - Lifetime
- 1986-02-06 AU AU55145/86A patent/AU590258B2/en not_active Expired
- 1986-02-06 DE DE8686901361T patent/DE3671245D1/de not_active Expired - Lifetime
-
1997
- 1997-10-17 US US08/953,217 patent/US20010018199A1/en not_active Abandoned
-
2003
- 2003-10-20 US US10/689,445 patent/US20040235090A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO8604609A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP0217814B2 (de) | 1994-12-21 |
US20040235090A1 (en) | 2004-11-25 |
US20010018199A1 (en) | 2001-08-30 |
DK55685D0 (da) | 1985-02-07 |
DK55685A (da) | 1986-08-08 |
WO1986004609A1 (en) | 1986-08-14 |
JPH0665318B2 (ja) | 1994-08-24 |
EP0217814B1 (de) | 1990-05-16 |
DE3671245D1 (de) | 1990-06-21 |
AU5514586A (en) | 1986-08-26 |
JPS62501609A (ja) | 1987-07-02 |
AU590258B2 (en) | 1989-11-02 |
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